To date, most clinical diagnostic assays for the detection of small quantities of analytes in fluids have been conducted as individual tests; that is, as single tests conducted upon single samples to detect individual analytes. More recently, multiple-sample preparation and automated reagent addition devices and multiple-sample assay devices, either in parallel or in serial procession, have been designed to improve efficiency and economy. Such automated reagent preparation devices and automated multiplex analyzers are often integrated into a single device.
Large-scale clinical laboratory analyzers of this type can accurately perform hundreds of assays in one hour automatically or semi-automatically. However, these analyzers are expensive and only centralized laboratories and hospitals can afford them. Such centralization necessitates sample transport to the laboratory or hospital, and often precludes urgent or emergent analysis of time-critical samples.
Thus, to address these problems, there is an increasing need for clinical analyzers which are cheap and easy-to-handle for everyone, such as clinical analyzers suitable for use at the patient bedside or in the patient's home without dedicated detectors. Blood glucose and pregnancy testers are well known examples.
Although useful tests of this sort have been offered for many years, a major breakthrough was the introduction of solid phase immunoassays and other strip tests since 1980. Most notable are Advance® test (Johnson & Johnson), TAM™ hCG assay (Monoclonal Antibodies, Inc.), Clear Blue Easy™ (Unipath Ltd.), and ICON (Hybritech). Commercially Available are Quantab™ (Environmental Test Systems), AccuLevel® (Syva), AccuMeter® (ChemTrak), Clinimeter™ (Crystal Diagnostics), and Q.E.D.™ (Enxymatics). One of the newest is a thermometer-type assay device (Ertinghausen G., U.S. Pat. No. 5,087,556) that is not yet commercially available. These systems can be used to assay blood levels of therapeutic drugs and general chemical analytes such as cholesterols.
One disadvantage, however, of each of these formats is that only one, or a very limited number, of assays can conveniently be performed simultaneously.
To fill the gap between massive analyzers and strip testers, some small instruments have been developed. The most notable is Eclipse ICA™ (Biotope, Inc.). This device is an automated centrifugal immunoassay and chemistry system. Patient samples are pipetted into cassettes that are placed into a rotary device. Sixteen tests can be run in approximately 17 minutes. The results are measured by UV/Nisible spectrometry or by fluorometry.
Despite these developments, there still exists a need for a simple device that can easily be used for multiple quantitative assays without a specialized detector.
<Spatially Addressable Probe Assays>
Recently, spatially addressable arrays of different biomaterials have been fabricated on solid supports. These probe arrays permit the simultaneous analysis of a large number of analytes. Examples are arrays of oligonucleotides or peptides that are fixed to a solid support and that capture complementary analytes. One such system is described by Fodor et al., Nature, Vol. 364, Aug. 5, 1993. Short oligonucleotide probes attached to a solid support bind complementary sequences contained in longer strands of DNA in liquid sample; the sequence of the sample nucleic acids is then calculated by computer based on the hybridization data so collected.
There remains a need for an economical system to fabricate spatially addressable probe arrays in a simplified format that provides both for ready detection and the ability to assay for large numbers of test substances (i.e. analytes) in a fluid test sample in a single step, or a minimum number of steps, or assay for a single test substance or analyte in a large number of fluid test samples.
<Spatially Addressable Laser-Based Detection Systems>
Several devices permit spatially addressable detection of digital information. In particular, several formats have been developed based on differential reflectance and transmittance of recording information.
In conventional audio or CD-ROM compact disks, digital information or digitally encoded analog information is encoded on a circular plastic disk by means of indentations in the disk. Typically, such indentations are on the order of one-eighth to one-quarter of the wavelength of the incident beam of a laser that is used to read the information from the disk. The indentations on the disk cause destructive interference within the reflected beam, which corresponds to a bit having a “zero” value. The flat areas of the disk reflect the laser beam back to a detector and the detector gives a value of “one” to the corresponding bit.
In another convention, a change of intensity of a reflected light beam gets a value of one while a constant intensity corresponds to zero.
Since the indentations have been formed in the disk in a regular pattern from a master copy containing a predetermined distribution of bits of “zero” and bits of “one”, the resultant signal received by the detector is able to be processed to reproduce the same information that was encoded in the master disk.
The standard compact disk is formed from a 12-cm polycarbonate substrate, a reflective metal layer, and a protective lacquer coating. The format of current CDs and CD-ROMs is described by the ISO 9660 industry standard.
The polycarbonate substrate is optical-quality clear polycarbonate. In a standard pressed, or mass-replicated CD, the data layer is part of the polycarbonate substrate, and the data are impressed in the form of a series of pits by a stamper during the injection molding process. The stamping master is typically glass.
Pits are continuously spirally impressed in the CD substrate. The reflective metal layer applied thereupon, typically aluminum, assumes the shape of the solid polycarbonate substrate, and differentially reflects the laser beam to the reading assembly depending on the presence or absence of “pits.” An acrylic lacquer is spin-coated as a thin layer on top of the reflective metal layer to protect it from abrasion and corrosion.
Although similar in concept and compatible with CD readers, the information is recorded differently in a recordable compact disk (CD-R). In CD-R, the data layer is separate from the polycarbonate substrate. The polycarbonate substrate instead has impressed upon it a continuous spiral groove as an address for guiding the incident laser. An organic dye is used to form the data layer. Cyanine or a metal-stabilized cyanine compound is generally used to form the data layer. An alternative material is phthalocyanine. One such metallophthalocyanine compound is described in U.S. Pat. No. 5,580,696.
In CD-R, the organic dye layer is sandwiched between the polycarbonate substrate and the metallized reflective layer, usually 24 carat gold, but alternatively silver, of the media. Information is recorded by a recording laser of appropriate preselected wavelength that selectively melts “pits” into the dye layer, causing the pits to become non-translucent. The reading sensor reads the presence or absence of pits from refractivity rather than differential reflectivity by physical pits in the standard CD. As in a standard CD, a lacquer coating protects the information layer.
Other physical formats for recording and storing information have been developed based on the same concept as the compact disk: creation of differential reflectance or transmittance on a substrate to be read by laser. One such format is termed digital versatile disk (DVD). A DVD looks like standard CD: it is a 120-mm (4.75 inch) disk with a hole in the center for engaging a rotatable drive mechanism. Like a CD, data is recorded on the disk in a spiral trail of tiny pits, and the disks are read using a laser beam. In contrast to a CD, which can store approximately 680 million bytes of digital data under the ISO 9660 standard, the DVD can store from 4.7 billion to 17 billion bytes of digital data. The DVD's larger capacity is achieved by making the pits smaller and the spiral tighter, that is, by reducing the pitch of the spiral, and by recording the data in as many as four layers, two on each side of the disk. The smaller pit size and tighter pitch require that the reading laser wavelength be smaller. While the smaller wavelength is compatible with standard pressed CDs, it is incompatible with current versions of the dye-based CD-R.
Thus, a single sided/single layer DVD can contain 4.7 GB of digital information. A single sided/dual layer DVD can contain 8.5 GB of information. A Dual sided/single layer disk can contain 9.4 GB of information, while a dual sided/dual layer DVD contains up to 17 GB of information.
Depending on the capacity, the disk may have one to four information layers. In the 8.5 GB and 17 GB options, a semi-reflector is used in order to access two information layers from one side of the disk. For the 8.5 GB DVD and 17 GB options, the second information layer per side may be molded into the second substrate or may be added as a photopolymer layer. In either case, a semi-reflector layer is required to allow both information layers to be read from one side of the disk. For the 17 GB DVD, it is necessary to produce two dual-layer substrates, and bond them together.
The DVD laser reader is designed to adjust its focus to either layer depth so that both of them can be quickly and automatically accessed.
All of the above-described formats require that the disk be spun. The nominal constant linear velocity of a DVD system is 3.5 to 4.0 meters per second (slightly faster for the larger pits in the dual layer versions), which is over 3 times the speed of a standard CD, which is 1.2 mps.
<Detection Method of DNA Chips>
DNA chips refer to chips having highly immobilized DNA probes of interest on solid substrates and are used for the analysis of a gene expression profile, genetic defects, etc., in samples. To investigate whether the sample contains a target nucleic acid that binds to the probe immobilized on the substrate, a detection system therefor is required.
Most currently-available genetic analysis DNA chips employ a method of fluorescently labeling a sample DNA, reacting it with the proves immobilized on the chip, and detecting the unreacted fluorescent material remaining on the chip surface using a confocal microscope or charge coupled device (CCD) imager (U.S. Pat. No. 6,141,096). However, such optical detection method is disadvantageous in size reduction and cannot display digitized outputs. For these reasons, research on the development of a new detection method for electrical signal outputs is actively being conducted.
Many research institutes, including Clinical Micro Sensors, are researching the electrochemical detection of DNA hybridization using a metal compound that is liable to oxidation/reduction (U.S. Pat. Nos. 6,096,273, 6,090,933). Separate compounds containing easily oxidizable/reducible metals form a complex upon DNA hybridization, and the complex is electrochemically detected (Anal. Chem., Vol., 70, pp. 4670-4677, 1998; J. Am. Chem. Soc., Vol. 119, pp. 9861-9870, 1997; Analytica Chemica Acta, Vol, Vo. 2886, pp. 216-224, 1994; Bioconjugate Chem., Vol. 8, pp. 906-913, 1997). However, this electrochemical method still needs separate labeling.
Approaches to assay methods not using the fluorescent label or any other labels have been actively made. As a result, a method of measuring a difference in mass before and after binding using a quartz crystal microbalance (Anal. Chem., Vol. 70, pp, 1288-1296, 1998), an assay method using matrix assisted laser description ionization (MALDI) mass spectrometry (Anal. Chem., Vol. 69, pp. 4540-4546, 1997, U.S. Pat. No. 6,043,031) were developed.
Even a single-base difference can be analyzed using a microfabricated cantilever, which is a mechanical sensor type for measuring molecular binding force before and after binding of DNA probe and target (Science, Vol., 288, pp. 316-318, 2000; Proc. Natl., Acad. Sci. USA, 98, 2560, 2001). However, this method needs additional equipment, such as a laser, for accurate measurement of cantilever beam deflection.
The present invention relates to the field of diagnosis and detection of small quantities of materials in fluids. It is an object of the present invention to provide cleavable signal elements using a cleavage technique specifically responsive to a double strand or single strand of nucleic acids or oligonucleotides having a complementary sequence, which are applicable to quantitative and qualitative assay devices, and a nucleic acid hybridization assay method and device using the cleavable signal element.
It is another object of the present invention to provide an accurate method and device of diagnosing a variety of diseases from both single nucleotide polymorphism (SNP) detection and gene expression profile obtained using the nucleic acid hybridization assay device.
An analytical apparatus based on the nucleic acid hybridization assay method and device using the cleavage technique can be modified to use the standard laser-based detection system, such as CD-ROM reader or DVD reader, and can be coupled to a detector including an optical device, an electrochemical device, or a capacitance and impedance measurement device. The analytical apparatus and method according to the present invention are useful in both detecting a number of individual analytes in a test sample and detecting a single analyte in a large number of separate samples.
It is still another object of the present invention is to provide an remote diagnostic system providing convenience to both patients and doctors by transmitting and receiving the information read from the analytical apparatus and digitalized as computer software, through an existing communication network, such as the Internet.